Composition having antifungal activity

ABSTRACT

The present invention provides a composition having an antifungal activity without depending on conventional compositions having anticandidal activity or combinations of such compositions. The composition comprises a complex of lysozyme bonded to chitosan. The composition according to the present invention is applicable to candidiasis of the skin and mucous membranes, in particular, oral candidiasis and vagina candidiasis affecting a large number of patients and can ameliorate the symptoms of these diseases, heal the same and prevent infection of the same. The composition comprises a complex of lysozyme, which has been widely used as a highly safe natural food additive, with a polysaccharide and, therefore, can reassure patients who use the same and ease their burden. The composition according to the present invention comprises a complex of lysozyme, which is a highly safe natural food additive, with a polysaccharide and, therefore, can be safely used by patients without considering any risk.

TECHNICAL FIELD

This invention relates to a novel composition having an antifungalactivity.

TECHNICAL BACKGROUND

One of the problems that may arise during the treatment of HIV andcancer as well as during organ transplants is the risk of infectioncaused by microorganisms. The development of antibiotics that fightbacterial infections is already well established. The appearance ofdrug-resistant bacteria such as MRSA and VRE is already causingproblems, however, more serious than these are infections caused byfungi. In particular, candidiasis caused by Candida albicans andaspergillosis caused by genus Aspergillus are large obstacles duringtransplant operations.

Yeasts and filamentous fungi are eukaryotes, and are referred to asfungi in comparison to bacteria, which are prokaryotes. Certain types offungi exhibit pathogenicity towards humans and animals, and are regardedas causative microorganisms of fungal infections. The pathogenicity ofthese fungi is generally weak, however, they sometimes cause severesymptoms in patients who already have lowered resistance.

Because the various illnesses generated by fungi have an enormous effecton the health of humans and animals, the development of new medicinesthat are useful in the treatment thereof is greatly desired. Moreover,the intrusion of filamentous fungi in houses as a result of condensationand the like, which is reflective of modern housing conditions, causessymptoms such as allergies and the like in humans, and has a deleteriouseffect on the health of humans and animals. The development of newantifungal agents is desired as an effective countermeasure against suchphenomena.

Here, preparations that inhibit Candida mycelial growth are described inPatent document 1 and Non-patent document 1.

In Patent document 1 it is disclosed that a synergistic effect inanticandidal activity against in vitro Candida mycelial growth isevident if capric acid (decanoic acid) is used in combination with anyone of geraniol, eugenol, or citral. Moreover, it is also disclosed inPatent document 1 that, in oral candidiasis models in mice, acombination of capric acid and ginger essential oil had the greatesteffect towards improving tongue symptoms.

Furthermore, it is disclosed in Non-patent document 1 that a synergisticeffect in anticandidal activity against in vitro Candida mycelial growthis evident if capric acid is used in combination with terpinen-4-ol.Moreover, it is also disclosed in Non-patent document 1 that, in oralcandidiasis models in mice, reliable therapeutic effects including areduction in the viable bacteria count were apparent when capric acidand terpinen-4-ol were used in combination.

However, the aforementioned Patent document 1 and Non-patent document 1disclose nothing more than the combined effect of using a medium chainfatty acid such as decanoic acid, which is known to have anticandidalactivity, with a terpene alcohol such as terpinen-4-ol, which, in thesame way, is also known to have anticandidal activity.

Furthermore, as is shown in Patent document 2, taking note of the factthat a complex of lysozyme and chitosan (30 kDa) has antibacterialproperties, applications of this complex to foodstuffs and cosmetics maybe considered. This complex is extremely safe, and imparts a stabilityto the lysozyme whose importance is emphasized by product design and thelike, so as to further broaden the antibacterial spectrum.

However, nothing apart from the fact that this complex has antibacterialactivity against Escherichia Coli K12 is disclosed, and anyantibacterial activity against fungi such as genus Candida and genusAspergillus has not been confirmed.

Documents of the Prior Art Patent Documents

-   [Patent document 1] Japanese Unexamined Patent Application (JP-A)    No. 2013-40156-   [Patent document 2] Japanese Unexamined Patent Application (JP-A)    No. 2005-187401

Non-Patent Documents

-   [Non-patent document 1] Kentaro NINOMIYA, et. al., “Effects of    Inhibitory Activity on Mycelial Growth of Candida albicans and    Therapy for Murine Oral Candidiasis by the Combined Use of    Terpinen-4-ol and Middle-chain Fatty Acid, Capric Acid”, Journal of    the Pharmaceutical Society of Japan, The Pharmaceutical Society of    Japan, Jan. 1, 2013, Vol. 133, Issue 1, pp. 133-140.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In contrast, the inventors of the present application performedstrenuous investigations in order to provide a novel composition havingan antifungal activity without having to rely on existing compositionshaving anticandidal activity or on combinations of such compositions. Inthe course of these investigations, the inventors of the presentapplication discovered that a complex formed by bonding lysozyme tochitosan exhibited antifungal activity against fungi such as Candida.

Means for Solving the Problem

Namely, a composition having an antifungal activity according to thepresent invention is characterized in comprising a complex formed bybonding lysozyme to chitosan. A Candida proliferation suppressing actionor a Malassezia fungi proliferation suppressing action are included inthis antifungal activity. In other words, this composition contains aCandida proliferation suppressing composition, a Malassezia fungiproliferation suppressing composition, or a Trichophyton proliferationsuppressing composition. Effects of this composition having anantifungal activity are described below.

It is desirable that a composition having an antifungal activity furthercomprises terpene alcohol or a fatty acid.

Examples of a terpene alcohol include geraniol, menthol, andterpinen-4-ol.

It is desirable that the fatty acid is a fatty acid having a carbonnumber of 8 to 12. Specifically, fatty acids such as caprylic acid(carbon number 8), capric acid (oxygen number 10), and lauric acid(carbon number 12) may be considered.

Effects of the Invention

According to the present invention, the effect of suppressing Candidainfections in skin and mucous membranes is achieved. Accordingly, aneffect of the present invention is that the present invention can beapplied to candidiasis in skin or mucous membranes, and, in particular,to oral candidiasis and vaginal candidiasis which afflict a large numberof patients, and can improve symptoms, cure diseases, and preventinfections that may arise therefrom. Moreover, Lysozyme is widely usedas an extremely safe, natural food additive, and a composition having anantifungal activity that uses a complex of lysozyme and a polysaccharideis able to reassure a patient who uses it and alleviate their suffering.

Additionally, oral candidiasis and vaginal candidiasis tend to readilyrecur. However, there are limits to the application of antifungal agentsdue to side effects and the risk of resistant bacteria appearing. Thepresent invention uses a complex of lysozyme, which is an extremelysafe, natural food additive, and a polysaccharide, and can therefore besafely used by a patient without risks such as those described aboveneeding to be considered.

Furthermore, by using this composition having antifungal activity as aMalassezia fungi proliferation suppressing composition, it is possibleto prevent and ameliorate seborrheic dermatitis which occurs on the skinof humans and animals. Additionally, by using this composition havingantifungal activity as a Trichophyton proliferation suppressingcomposition, it is possible to prevent and ameliorate Trichophytoninfections such as athletes foot which occur on the skin of humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing Candida growth rates when a lysozyme-chitosancomplex is used in combination with terpinen-4-ol.

FIG. 2 is a view showing Candida growth rates when a lysozyme-chitosancomplex is used in combination with decanoic acid.

FIG. 3 is a view showing results when a viable bacteria count inside anoral cavity that is obtained when a lysozyme-chitosan complex is used incombination with terpinen-4-ol is measured in an oral candidiasis modelin mice.

FIG. 4 is a view showing a tongue symptom score obtained when alysozyme-chitosan complex is used in combination with terpinen-4-ol inan oral candidiasis model in mice.

FIG. 5 is a view showing Candida growth rates when lysozyme is used incombination with terpinen-4-ol.

FIG. 6 is a view showing Candida growth rates when chitosan is used incombination with terpinen-4-ol.

FIG. 7 is a view showing Candida growth rates when a lysozyme-chitosanblend is used in combination with terpinen-4-ol.

FIG. 8 is a view showing Candida growth rates when lysozyme is used incombination with decanoic acid.

FIG. 9 is a view showing Candida growth rates when chitosan is used incombination with decanoic acid.

FIG. 10 is a view showing Candida growth rates when a lysozyme-chitosanblend is used in combination with decanoic acid.

FIG. 11 is a view showing Candida growth rates at various concentrationsof simple lysozyme, simple chitosan, a lysozyme-chitosan blend, and alysozyme-chitosan complex.

FIG. 12 is a photograph showing a zone of inhibition of alysozyme-chitosan complex against Malassezia fungi.

FIG. 13 is a photograph showing states after seven days of colonyformation of Malassezia fungi in respective test samples.

FIG. 14 is a photograph showing states after seven days of colonyformation of Malassezia fungi in respective test samples.

FIG. 15 is a photograph showing states after seven days of colonyformation of Trichophyton in respective test samples.

FIG. 16 is a photograph showing states after seven days of colonyformation of Trichophyton in respective test samples.

EMBODIMENTS FOR IMPLEMENTING THE INVENTION

Hereinafter, the present invention will be described in detail.

1. First Embodiment

A composition having an antifungal activity according to a firstembodiment contains a complex formed by bonding lysozyme to chitosan (inwater-soluble form having a molecular weight of not more than 25000 Da).Note that, in the present embodiment, chitosan having a molecular weightof 14000 Da is used. Either chicken-derived lysozyme or human-derivedlysozyme may be used.

The lysozyme-chitosan complex of the present invention can bemanufactured by bonding lysozyme and chitosan together via a Maillardreaction. Because bonding the lysozyme and chitosan together via aMaillard reaction causes most of or all of the antigenic structure inthe lysozyme to be masked, even if there is some absorption of thelysozyme-chitosan complex, it is still difficult for this to cause anallergy.

The specific manufacturing method is as follows.

Quantities of lysozyme and chitosan are dissolved in water such that thelysozyme/chitosan mass ratio is more preferably from 60/40 to 40/60, andthe total content of lysozyme and chitosan in the aqueous solution isadjusted so as to be from 5 to 30% by mass. The resulting aqueoussolution is then freeze-dried so as to be changed into powder form. Thisobtained powder is then made to undergo a Maillard reaction for a periodof 2 to 20 days, and more preferably a period of 7 to 14 days inconditions of a temperature of 55 to 80° C., and more preferably atemperature of 55 to 65° C., and a relative humidity of 50 to 80%, andmore preferably a relative humidity of 60 to 70%. By performing thisprocess the lysozyme-chitosan complex of the present invention can bemanufactured.

In the formation of the lysozyme-chitosan complex of the presentinvention, the formation of a polymer material in the form of aprotein-chitosan complex can be confirmed using a plate obtained via SDS(sodium dodecyl sulfate-polyacrylamide) electrophoresis that hasundergone dyeing processing.

The composition having an antifungal activity of the present inventionwill now be described.

In order to have a greater antibacterial activity, the compositionhaving an antifungal activity of the present invention preferablycontains 10 to 100% by mass of the lysozyme-chitosan complex, and morepreferably 30 to 100% by mass thereof, and most preferably 50 to 100% bymass thereof.

Note that constituents such as dextrin and lecithin and the like can beused in addition to the lysozyme-chitosan complex in the compositionhaving an antifungal activity. The content of these other constituentspresent in addition to the lysozyme-chitosan complex in the compositionhaving an antifungal activity is preferably 0 to 90% by mass, morepreferably 0 to 70% by mass, and most preferably 0 to 50% by mass.

2. Second Embodiment

A composition having an antifungal activity according to a secondembodiment contains a complex formed by bonding lysozyme to chitosan viaa Maillard reaction, together with a terpene alcohol. Note that examplesof a terpene alcohol include geraniol, menthol, and terpinen-4-ol, andthe like. These terpene alcohols are plant essential oil components, andit is also possible to use plant essential oils that contain theseterpene alcohols.

3. Third Embodiment

A composition having an antifungal activity according to a thirdembodiment contains a complex formed by bonding lysozyme to chitosan viaa Maillard reaction, together with a fatty acid. Note that examples of afatty acid include caprylic acid (carbon number 8), capric acid (oxygennumber 10), and lauric acid (carbon number 12).

EXAMPLES

The present invention will now be described in further detail withexamples being provided, however, the present invention is not limitedsolely to these examples. Note that, in the following examples, alysozyme-chitosan complex may also be referred to as LYZOX (RegisteredTrademark of Wako Filter Technology Co. Ltd.).

[Example 1] Inhibitory Activity of LYZOX, terpinen-4-ol, and DecanoicAcid Against Candida Mycelial Growth

Methods of performing an in vitro inhibition test for yeast-form andmycelial growths of Candida are well-established, and as a result, theMIC (minimum inhibitory concentration) of various types of material canbe measured.

A clinical isolate, Candida albicans TIMM 1768, held by the TeikyoUniversity Medical Mycology Research Center was used as the Candida.This Candida was cultured for 20 hours at 37° C. on a Sabouraud DextroseAgar culture plate. The multiplied bacterial cells were recovered, andthen suspended in sterilized water so as to form Candida solutions whosebacterial count was adjusted to 5×103 cells/ml. Each sample wasdissolved in advance in DMSO (dimethyl sulfoxide) so as to match thefinal concentration, and was then added to a ⅓ RPMI-1640 culture mediumcontaining 2% calf serum so as to form sample solutions. 100 μL of boththe sample solution (having a DSMO concentration in the culture mediumof 0.5%) and the Candida bacterial solution were then placedrespectively in each well of a 96-hole microplate (manufactured bySumitomo Bakelite, Tokyo), and these were then cultured for three hoursat 37° C. in a 5% carbon dioxide gas atmosphere. After three hours, thesolutions in each well were discarded and the wells were cleaned.Subsequently, 200 μL of ⅓ RPMI-1640 was newly added to each well, andculturing was performed for 16 hours at 37° C. in a presence of 5%carbon dioxide gas. Once this culturing had ended, the solutions in eachwell were removed by suction, and the wells were washed usingphysiological saline. 200 μL of 70% ethanol was then injected so as tosterilize the Candida. After the ethanol had been removed, and the wellswashed with tap water, 100 μL of a dye solution (i.e., a 0.1 M phosphatebuffer containing 0.01% of dissolved crystal violet solution) wasinjected and left standing for 15 minutes. As a result, the Candidamycelia adhering to the surface of the wells were dyed.

After the wells were then washed with tap water so as to remove excessdye solution, 150 μL of 3-isopropanol containing 0.04 NHCL, and 50 μL ofa 0.25% sodium dodecyl sulfate solution were then injected so as to freethe coloring adhering to the bacterial cells. After the coloring hadbeen freed, the plate was set in a multiscan photometer (i.e., aMultiskan FC, manufactured by Thermo Fisher Scientific Inc.), and the ODat 620 nm was measured in each well. The growth inhibition rate was thendetermined using the following formula.

Growth inhibition rate (%)=(1—sample OD/subject OD)×100

As is shown in FIG. 1 and FIG. 2, an IC 70 (i.e., a 70% growthinhibitory concentration) of each of the LYZOX, terpinen-4-ol, anddecanoic acid were as follows.

LYZOX (alone) 193 μg/ml Terpinen-4-ol (alone) 1900 μg/ml  Capric acid(Decanoic acid) (alone) 480 μg/ml

[Example 2] Synergistic Effect in Anticandidal Activity Against CandidaMycelial Growth Obtained from Combination of LYZOX with terpinen-4-ol orDecanoic Acid

Generally, the definition of a synergistic effect when antibacterialsubstances are used in combinations against microorganisms is an effectthat is significantly greater than the sum of the individual effects oftwo agents. As is described below, the evaluation of a synergisticeffect can be performed by means of a checkerboard method using an FICindex (Fractional Inhibitory Concentration index) determined from theMIC values of two agents. Namely, it is determined that a synergisticeffect exists when the FIC index is less than 0.5.

(How to Determine an FIC Index)

FIC index=A ₁ /A ₀ +B ₁ /B ₀

A₀: MIC of A agent alone

A₁: MIC of A agent when A agent is used in combination with B agent

B₀: MIC of B agent alone

B₁: MIC of B agent when A agent is used in combination with B agent

(Evaluation of Effect of Combined Use Obtained from FIC Index)

FIC < 0.5 Synergistic effect 0.5 < FIC < 1.0 Extreme synergistic effectFIC = 1.0 Synergistic effect 1.0 < FIC = 2.0 N/A FIC > 2.0 Antagonisticaction

The anticandidal activity synergistic effect provided by the combineduse was evaluated using a checkerboard method. A hypotheticalcheckerboard was placed on top of the 96-hole microplate, andconcentration series of each of the terpinen-4-ol or decanoic acid andthe LYZOX were made to mutually intersect. The experiment conditions andthe measurement method and the like were the same as those employed forthe mycelial growth test (i.e., Example 1).

When LYZOX was used in combination with terpinen-4-ol, as is shown inFIG. 1, the IC (at a 70% growth inhibitory concentration) of the LYZOXand terpinen-4-ol were as follows.

LYZOX (used in combination)  60 μg/ml Terpinen-4-ol (used incombination) 280 μg/ml

In other words, when LYZOX and terpinen-4-ol were used in combination,the FIC index with respect to the mycelial growth of the Candida was0.447, and it was therefore determined that a synergistic effectexisted.

When LYZOX was used in combination with capric acid (decanoic acid), asis shown in FIG. 2, the IC (at a 70% growth inhibitory concentration) ofthe LYZOX and capric acid (decanoic acid) were as follows.

LYZOX (used in combination) 30 μg/ml Capric acid (Decanoic acid) (usedin combination) 25 μg/ml

In other words, when LYZOX and terpinen-4-ol were used in combination,the FIC index with respect to the mycelial growth of the Candida was0.208, and it was therefore determined that a synergistic effectexisted.

[Example 3] Therapeutic Effect Obtained from Combination of LYZOX withterpinen-4-ol in an Oral Candidiasis Model in Mice

Using ICR strain mice (males, 6 weeks old, Charles River Laboratories,Japan) as the experimental animals, these mice were subcutaneouslyinjected with 100 mg/kg of prednisolone as an immunosuppressant on theday prior to the candidiasis inoculation. Additionally, tap watercontaining 15 mg/ml of chlortetracycline hydrochloride was made freelyavailable starting from that same day. On the day of the innoculation,in order to keep the mice in a resting state, 14.4 mg/kg ofchlorpromazine hydrochloride (manufactured by Wako Pure ChemicalIndustries, Ltd.) was intramuscularly administered in advance. Thebacterial strain and culturing method that were used were the same as inthe Examples as far as the previous paragraph, and the multipliedbacterial cells were suspended in an RPMI-1640 culture medium containing2% calf serum so as to form a bacterial solution whose bacterial countwas adjusted to 2×10⁸ cells/ml. A cotton bud was then soaked in thisbacterial solution, and then rubbed inside the oral cavity of theresting mice so as to inoculate the mice with the Candida bacteria.

The samples in the table below were administered to the oral candidiasismice in the following manner. Firstly, the samples were suspended inadvance in distilled water using Tween 80 (having a final concentrationof 1%), and then 3, 24, and 27 hours after the candidiasis inoculation,using a gastric tube for mice, droplets thereof were administered ontothe tongue dorsum inside the oral cavity. Two days after the inoculationthe mouse was euthanized, and the tongue symptom score was evaluated inaccordance with a reference. The interior of the oral cavity of eachmouse was then wiped with a cotton bud, and the Candida bacterial cellsrecovered by that cotton bud were suspended in a saline solution. Afixed quantity of this was then coated onto a Candida GS agar plate, andwas cultured at 37° C. for 20 hours. The number of colonies appearingafter this time was then measured. The number of viable Candida bacteriaCFU (colony forming units) recovered from the relevant individuals wasthen calculated from the number of colonies.

TABLE 1 Inoculation Number No. Group Infection sample of mice 1 Negativecontrol group + FLCZ 10 mg/ml 3 2 Positive control group + Water 3 3LYZOX only group + L 20 mg/ml 3 4 Terpine-4-ol only + T 10 mg/ml 3 group5 LYZOX + Terpine-4-ol + 10 mg/ml 3 group 6 Non-infected group − Water 3

As is shown in FIG. 3, the results of this experiment showed that areduction in the viable bacterial count in the terpinen-4-ol only groupwas recognized for the viable bacteria count recovered from the oralcavity. Moreover, as is shown in FIG. 4, a remarkable improvement effectwas recognized in the tongue symptom score in the LYZOX only group.

[Example 4] Anticandidal Activity Effect Obtained from Simple Lysozyme,Simple Chitosan, a Lysozyme-Chitosan Blend, and a Combination of LYZOXwith terpinen-4-ol Against Candida Mycelial Growth

The anticandidal activity synergistic effect obtained from a combineduse was evaluated using a checkerboard method in the same way as inExample 2. A hypothetical checkerboard was placed on top of a 96-holemicroplate, and concentration series of each of the simple lysozyme, thesimple chitosan, the lysozyme-chitosan blend, and the combination ofLYZOX with terpinen-4-ol were made to mutually intersect. The experimentconditions and the measurement method and the like were the same asthose employed for the mycelial growth test (i.e., Example 1).

As is shown in FIG. 5 through FIG. 7, the experiment results showed thatwhen simple lysozyme was used in combination with terpinen-4-ol, whensimple chitosan was used in combination with terpinen-4-ol, and when amixture of lysozyme with chitosan was used in combination withterpinen-4-ol, then no improvement effect in the anticandidal activityobtained from a combined usage against Candida mycelial growth was seen.In contrast, as is shown in FIG. 1, when LYZOX was used in combinationwith terpinen-4-ol, then compared with the three patterns of combineduse described above, a remarkable improvement effect was recognized.

[Example 5] Anticandidal Activity Effect Obtained from Simple Lysozyme,Simple Chitosan, a Lysozyme-Chitosan Blend, and a Combination of LYZOXwith Decanoic Acid Against Candida Mycelial Growth

The anticandidal activity synergistic effect obtained from a combineduse was evaluated using a checkerboard method in the same way as inExample 2. A hypothetical checkerboard was placed on top of a 96-holemicroplate, and concentration series of each of the simple lysozyme, thesimple chitosan, the lysozyme-chitosan blend, and the combination ofLYZOX with decanoic acid were made to mutually intersect. The experimentconditions and the measurement method and the like were the same asthose employed for the mycelial growth test (i.e., Example 1).

As is shown in FIG. 8 through FIG. 10, the experiment results showedthat when simple lysozyme was used in combination with decanoic acid,when simple chitosan was used in combination with decanoic acid, andwhen a mixture of lysozyme with chitosan was used in combination withdecanoic acid, then no improvement effect in the anticandidal activityobtained from a combined usage against Candida mycelial growth was seen.In contrast, as is shown in FIG. 2, when LYZOX was used in combinationwith decanoic acid, then compared with the three patterns of combineduse described above, a remarkable improvement effect was recognized.

[Example 6] Anticandidal Activity Effect Obtained from Simple Lysozyme,Simple Chitosan, a Lysozyme-Chitosan Blend, and LYZOX Against CandidaMycelial Growth

The anticandidal activity effect obtained from each test reagent wasevaluated using a checkerboard method in the same way as in Example 2. Ahypothetical checkerboard was placed on top of a 96-hole microplate, andconcentration series of each of the simple lysozyme, the simplechitosan, the lysozyme-chitosan blend, and the LYZOX were prepared. Theexperiment conditions and the measurement method and the like were thesame as those employed for the mycelial growth test (i.e., Example 1).

As is shown in FIG. 11, the experiment results showed that when simplelysozyme was used and when simple chitosan was used, no improvementeffect in the anticandidal activity against Candida mycelial growth wasseen. Moreover, when a mixture of lysozyme with chitosan (MIX) was used,although an improvement effect in the anticandidal activity againstCandida mycelial growth was visible, it was seen that the anticandidalactivity effect gradually reduced as the concentration increased. Incontrast, when LYZOX was used, an improvement effect in the anticandidalactivity against Candida mycelial growth was visible, and it was seenthat the anticandidal activity effect gradually increased as theconcentration increased. In particular, when the LYZOX concentration was250 μg/ml, the growth rate dropped to almost zero (actually, 0.9%).

[Example 7] Anti-Malassezia Fungal Activity Effect Obtained fromKetoconazole, and from a Complex of Lysozyme and Chitosan AgainstMalassezia Fungi (Malassezia pachydermatis)

In the Malassezia sensitivity test, 100 μL lots of a test bacterialsolution adjusted to McFarland 1.0 were coated over an Agar plate usingan MLNA culture medium (the composition of which is shown in Table 2).Sterile paper disks having a paper thickness of 8 mm which are used forinspecting antibiotics were placed on a Petri dish, and 50 μL of eachtest sample was dripped onto these paper disks. After the test sampleshad subsequently been cultured for seven days at 32° C., they wereobserved and their properties were determined. In addition, the diameterof the zone of inhibition was measured. Note that 0.5% DMSO,ketoconazole 10 μg/ml, ketoconazole 100 μg/ml, LYZOX 5 mg/ml, LYZOX 10mg/ml, LYZOX 20 mg/ml, and LYZOX 40 mg/ml were used as the test samples.

MLNA Culture medium 25 ml/plate

TABLE 2 Bacto Peptone 5 g Glucose 5 g Yeast extract 1 g Oxbiledesiccated 4 g Glycerol 5 ml Glycerol monostearate 0.25 g Tween60 2.5 mlAgar 7.5 g DW 500 ml Chloramphenicol 0.0125 g

The test samples were placed in an autoclave at 110° C. for 20 minutes.

Results for the Malassezia zone of inhibition (mm) of each test sampleare shown below.

TABLE 3 Diameter of zone of inhibition (mm) Test sample (50 μl) Exp. 1Exp. 2 DW 0 0 0.5% DMSO 0 0 Ketoconazole 10 μg/ml 11 × 12 — Ketoconazole100 μg/ml 27 × 28 24 × 24 LYZOX 5 mg/ml — 10 × 11 LYZOX 10 mg/ml 16 × 1614 × 15 LYZOX 20 mg/ml 21 × 21 — LYZOX 40 mg/ml 24 × 24 —

As is shown in FIG. 12, in Exp. 1, a zone of inhibition of 16×16 mm (ona paper disk of 8 mm) was visible around the paper disk onto which 50 μlof LYZOX 10 mg/ml had been dripped. In the same way, a zone ofinhibition of 21×21 mm was visible around the paper disk onto which 50μl of LYZOX 20 mg/ml had been dripped, and a zone of inhibition of 24×24mm was visible around the paper disk onto which 50 III of LYZOX 40 mg/mlhad been dripped. No zone of inhibition was visible for the sterilizedwater of the control.

In Exp. 2, a zone of inhibition of 14×15 mm was visible around the paperdisk onto which 50 μl of LYZOX 10 mg/ml had been dripped, and a zone ofinhibition of 10×11 mm was visible around the paper disk onto which 50μl of LYZOX 5 mg/ml had been dripped.

Example 8

The anti-Malassezia fungal activity obtained from LYZOX againstMalassezia fungi (Malassezia pachydermatis) was evaluated using an agardilution method. Specifically, an agar plate (having the samecomposition as that described above in Table 2) was prepared using MLNAculture mediums to which the LIZOX and other samples had been added soas to attain the respective concentrations (final concentrations) givenin Table 4 (see below). 100 μL lots of the test bacterial solutionadjusted to McFarland 1.0 were coated over this Agar plate and werecultured for seven days at 32° C. Thereafter, the growth rate of thebacteria thereon were compared with a control (i.e., an MLNA culturemedium containing no test additives) and their properties weredetermined.

Results for the negativity and positivity of the bacteria obtained fromeach test sample are shown below. Note that if the mycelial growth rateof the control is taken as 100%, then when the mycelial growth rate inthe test sample is 0%, this is determined to be [−], while if themycelial growth rate in the test sample is 1 to 20%, this is determinedto be [+], and if the mycelial growth rate in the test sample is 21 to90%, this is determined to be [+++]. The state of colony formation ofthe bacteria in each test sample is shown in FIG. 11 and FIG. 12. Notethat FIG. 13 and FIG. 14 are photographs showing states 7 days aftercolony formation of the Malassezia fungi in each test sample.

TABLE 4 Concentration Sample name (mg/ml) Determination LYZOX 1 − 0.5 −0.1 ++ 0.05 +++ Decanoic acid 2 + 1 +++ 0.2 +++ 0.1 +++ LYZOX/ 1/2 −Decanoic acid 0.5/1   − 0.1/0.2 ++ 0.05/0.1  ++ Lysozyme 0.5 +++ 0.05+++ Chitosan 5 − 0.05 − Lysozyme/ 0.5/0.5 − Chitosan 0.25/0.25 −0.05/0.05 ++ 0.025/0.025 ++ Mycelial growth rate taking control as 100Determination 0% − 1 to 20% + 21 to 90%  ++ 91 to 100% +++

From the results of this experiment the growth inhibitory concentrationsin each sample were found to be as follows.

TABLE 5 Sample name Growth inhibitory concentration (mg/ml) LYZOX 0.5 Decanoic acid — LYZOX/Decanoic acid 0.5/1   Lysozyme — Chitosan 0.05Lysozyme/Chitosan 0.5/0.5

From the above-described experiments, it was found that LYZOX aloneexhibited anti-Malassezia fungal activity against Malassezia fungi.Furthermore, it was found that LYZOX used in combination with decanoicacid also exhibited anti-Malassezia fungal activity, however, noimprovement effect compared with the LYZOX alone was found. It may beconsidered that the fact that Malassezia fungi are a perfect yeast typeof fungus causes the anti-bacterial effects thereof to be different fromthose of other types of fungus.

[Example 9] Anti-Trichophyton Activity of LYZOX and Decanoic AcidAgainst Trichophyton, and Synergistic Effect of Anti-TrichophytonActivity Obtained by Using these in Combination

In the same way as in the test for Candida, methods of performing an invitro inhibition test for Trichophyton are well-established, and as aresult, the MIC (minimum inhibitory concentration) of various types ofmaterial can be measured.

A clinical isolate, Trichophyton mentagrophytes TIMM 2789, held by theTeikyo University Medical Mycology Research Center was used as theTrichophyton. For the preculture process, this Trichophyton wasextracted from low-temperature silica stock one week prior to the day ofthe test, and was cultured on a Sabouraud Dextrose Agar culture platecontaining 50 μg/ml of chloramphenicol and 500 μg/ml of cycloheximide(such that these components were diluted to 1/10). This culturing wasperformed for one week at 28° C., and the bacteria were collected andused on the day of the test. The setting of the Trichophyton bacterialcount was achieved by transferring the collected bacteria using apour-plate method to a culture medium such that the final concentrationwas 10³ cells/ml. In addition, the respective test samples were alsotransferred to a culture medium using a pour-plate method such that thefinal concentrations thereof were set to those shown in the table below.An RPMI culture medium (diluted to a final concentration of ⅓)containing a bacterial solution adjusted with 1.5% agar was also held at37° C. so that it did not solidify during the preparation process. Inthe preparation, 1 ml of each test sample solution and 24 ml ofRPMI-1640 culture medium were blended together, and prepared in asterilized plastic Petri dish. The Trichophyton inoculation culturemedium that was prepared in this manner was then cultured at 28° C. forfour days, and the growth inhibitory effect of the mycelia after thisculturing was observed and the properties thereof determined. Thedeterminations were made both visually, and in the case of turbiditybeing present, by comparing the growth condition of the mycelia with acontrol (containing no test sample additives) via photomicrograph (atmagnification ×40).

Results for the negativity and positivity of the bacteria obtained fromeach test sample are shown below in Table 6. Note that if the mycelialgrowth rate of the control is taken as 100%, then when the mycelialgrowth rate in the test sample is 0%, this is determined to be [−],while if the mycelial growth rate in the test sample is 1 to 20%, thisis determined to be [+], and if the mycelial growth rate in the testsample is 21 to 90%, this is determined to be [+++]. The state of colonyformation of the bacteria in each test sample is shown in FIG. 11 andFIG. 12. Note that FIG. 15 and FIG. 16 are photographs showing states 7days after colony formation of the Trichophyton in each test sample.

TABLE 6 Concentration Sample name (mg/ml) Determination LYZOX 10 − 5 ++2.5 ++ 1 +++ Decanoic acid 1.5 − 1 ++ 0.9 +++ 0.8 +++ LYZOX/ 0.3/0.6 −Decanoic acid 0.2/0.4 − 0.1/0.2 − 0.05/0.1  ++ Lysozyme 5 ++ 2.5 +++Chitosan 5 ++ 2.5 +++ Lysozyme/ 5/5 ++ Chitosan 2.5/2.5 ++ Lysozyme/0.25/0.25/1 − Chitosan/ 0.2/0.2/0.8 ++ Decanoic acid 0.05/0.05/0.2 ++0.025/0.025/0.1 ++ Mycelial growth rate taking control as 100Determination 0% − 1 to 20% + 21 to 90%  ++ 91 to 100% +++

The MIC (minimum inhibitory concentration) of the LYZOX and the decanoicacid were as follows.

LYZOX (used alone)  10 mg/ml Decanoic acid (used alone) 1.5 mg/ml

When LYZOX and decanoic acid were used in combination, the MIC (minimuminhibitory concentration) of the LYZOX and the decanoic acid were asfollows.

LYZOX (used in combination) 0.1 mg/ml Decanoic acid (used incombination) 0.2 mg/ml

Namely, when LYZOX and decanoic acid were used in combination, the FICindex with respect to growth suppression of the Trichophyton was 0.143,and it was therefore determined that a synergistic effect existed.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to provide a novelcomposition having an antifungal activity without having to rely onexisting compositions having an antifungal activity including activityagainst Candida or on combinations of such compositions.

1. An antifungal comprising a complex formed by bonding lysozyme to chitosan.
 2. The antifungal according to claim 1, wherein the antifungal has an antifungal activity selected from the group consisting of a Candida proliferation suppressing action, a Malassezia fungi proliferation suppressing action, and a Trichophyton proliferation suppressing action.
 3. The antifungal according to claim 1, wherein the antifungal further includes terpene alcohol or a fatty acid.
 4. The antifungal according to claim 3, wherein the terpene alcohol is terpinen-4-ol, hinokitiol, menthol, or geraniol.
 5. The antifungal according to claim 3, wherein the fatty acid is a fatty acid having a carbon number of 8 to
 12. 6. The antifungal according to claim 3, wherein the fatty acid is capric acid or lauric acid.
 7. The antifungal according to claim 1, wherein the antifungal further comprises decanoic acid.
 8. The antifungal according to claim 7 for suppressing a Trichophyton proliferation.
 9. A method for suppressing a fungus by applying a complex formed by bonding lysozyme to chitosan.
 10. The method according to claim 9, wherein the suppression of the fungus is caused by an antifungal activity selected from the group consisting of a Candida proliferation suppressing action, a Malassezia fungi proliferation suppressing action, and a Trichophyton proliferation suppressing action.
 11. The method according to claim 9 further applying terpene alcohol or a fatty acid.
 12. The method according to claim 11, wherein the terpene alcohol is terpinen-4-ol, hinokitiol, menthol, or geraniol.
 13. The method according to claim 11, wherein the fatty acid is a fatty acid having a carbon number of 8 to
 12. 14. The method according to claim 11, wherein the fatty acid is capric acid or lauric acid.
 15. The method according to claim 9, further applying decanoic acid.
 16. The method according to claim 15, wherein the suppression of the fungus is caused by an antifungal activity of a Trichophyton proliferation suppressing action.
 17. A method for preparing an antifungal wherein the method comprises a step of bonding lysozyme to chitosan to form a complex, and a step of preparing the antifungal by containing the complex.
 18. The method according to claim 17, wherein the antifungal further includes terpene alcohol or a fatty acid.
 19. The method according to claim 17, wherein the antifungal further comprises decanoic acid.
 20. The method according to claim 19, wherein the antifungal is for suppressing a Trichophyton proliferation. 